ABSTRACT

Metallo-β-lactamase enzymes (MβL) are encoded by transferable genes, which appear to spread rapidly among gram-negative bacteria. The objective of this study was to develop a multiplex real-time PCR assay followed by a melt curve step for rapid detection and identification of genes encoding MβL-type enzymes based on the amplicon melting peak. The reference sequences of all genes encoding IMP and VIM types, SPM-1, GIM-1, and SIM-1 were downloaded from GenBank, and primers were designed to obtain amplicons showing different sizes and melting peak temperatures (Tm). The real-time PCR assay was able to detect all MβL-harboring clinical isolates, and the Tm-assigned genotypes were 100% coincident with previous sequencing results. This assay could be suitable for identification of MβL-producing gram-negative bacteria by molecular diagnostic laboratories.

The prevalence of MβL-producing gram-negative bacilli has increased in some hospitals, particularly among clinical isolates of Pseudomonas aeruginosa and Acinetobacter spp. (21, 23, 27). Since MβL production may confer phenotypic resistance to virtually all clinically available β-lactams, the continued spread of MβL is a major clinical concern (26). The aim of this study was to develop a multiplex real-time PCR assay followed by a melt curve step for rapid detection and identification of genes encoding the MβL-type enzymes so far described. The MβL type identification was based on the characteristic amplicon melting peak.

MATERIALS AND METHODS

MβL-harboring clinical isolates and MβL-negative control strains.The strains used in this study are listed in Tables 1 and 2. The MβL genotypes of the clinical isolates of gram-negative nonfermentative and fermentative bacteria harboring MβL were previously characterized by PCR and sequencing. When applicable, these clinical isolates were also previously molecularly typed to ensure that genetically unrelated strains were used. Additionally, several American Type Culture Collection (ATCC; Manassas, VA) reference strains and laboratory strains were used as MβL-negative controls (Table 2).

ATCC reference and laboratory strains of gram-negative bacteria used during the validation process as MβL-negative control strainsa

DNA preparation.The microorganisms were grown on blood agar plates overnight at 37°C to ensure colony purity. Three or four bacterial colonies were taken from the blood agar plates and suspended in 200 μl of DNase/RNase-free distilled water (Invitrogen, CA). Two microliters of this suspension was used as templates for further amplification.

Primer design.The currently available reference sequences of the MβL-encoding IMP- and VIM-type (http://www.lahey.org/studies/), SPM-1 (AJ492820), GIM-1 (AJ620678), and SIM-1 (AY887066) genes were downloaded from GenBank (National Center for Biotechnology Information, National Institutes of Health, Bethesda, MD). Based on the comprehensive analyses and alignments of each MβL type, primers were designed to yield amplicons showing different sizes and melting peak temperatures (Tm) separated by at least 1°C. Predicted amplicon sizes and Tm were determined by the Lasergene software package (DNASTAR, Madison, WI).

Additionally, a primer pair targeting the consensus region of the bacterial 16S rRNA gene was included in the reaction mixture as a PCR internal-control target. Primer pairs were evaluated in a single format (using a primer concentration of 0.5 μM) to ensure that they correctly amplified their respective loci and that the amplicons showed the expected Tm. Subsequently, the multiplex format was optimized by assaying different primer pair concentrations. The primer sequences, positions, and concentrations, and the sizes of the corresponding amplicons, are given in Table 3.

Multiplex real-time PCR validation.In order to assess the accuracy of the assay, 44 bacterial strains were blindly tested after real-time PCR optimization (Tables 1 and 2).

Multiplex real-time PCR sensitivity.The sensitivity of the reaction was estimated by dilution experiments. Briefly, one representative of each MβL-harboring clinical isolate was suspended in DNase/RNase-free distilled water to a density corresponding to a McFarland turbidity standard of 1.0 (3 × 108 CFU/ml). These suspensions were used to prepare serial 10-fold dilutions using DNase/RNase-free distilled water.

RESULTS AND DISCUSSION

When different strains were submitted to the real-time PCR assay, differences in the Tm of the amplicons were observed for strains harboring blaIMP-type allelic variants (from 76.0°C to 77.5°C) as well as for those harboring blaVIM-type allelic variants (from 87.5°C to 88.5°C) (Table 1). These differences in Tm will be observed mainly for amplicons generated from blaIMP-type genes, since the GC contents of the amplicons generated will be more divergent than those for blaVIM-type genes (Table 1).

Allelic variants for the remaining MβL types (blaSPM-1, blaGIM-1, and blaSIM-1) have not been found yet. For this reason, only one clinical isolate harboring blaGIM-1 and one harboring blaSIM-1 were used during the validation process. The theoretical and practical Tm obtained were very similar, and no Tm differences were observed when several genetically unrelated blaSPM-1-harboring P. aeruginosa isolates were submitted to the assay (Table 1).

When the negative-control ATCC reference strains and laboratory strains were submitted to the assay, the melt curve analysis showed only one melting peak varying from 85.5°C to 86.5°C (Table 2). These melting peaks were consistent with the Tm of the amplicon generated by the primers targeting the conserved sequences of the 16S rRNA gene. This internal-control primer pair was used at a lower concentration than the primers targeting the MβL genes; thus, the latter would have preference during the amplification reaction. This strategy was employed to avoid double amplification, which could compromise the melt curve analysis.

The real-time PCR sensitivity experiment showed that the assay was capable of detecting the16S rRNA target gene at a dilution corresponding to 6 × 103 CFU per reaction; blaSPM, blaVIM, blaSIM, and blaIMP at 6 × 102 CFU per reaction; and blaGIM at 6 × 101 CFU per reaction (data not shown). Additionally, the lowest detection limits of the target genes were represented by the cycle threshold values of 34.62, 34.11, 33.66, 32.69, 32.58, and 28.86 for the16S rRNA, blaSPM, blaGIM, blaIMP, blaSIM, and blaVIM genes, respectively. This suggests that the assay as developed is sufficiently robust, even when the bacterial cells suspended in water are used as the template.

Although the assay was developed to detect all MβL-encoding genes, we could not submit strains harboring all the blaIMP and blaVIM allelic variants, since we do not have access to all of them. We also acknowledge the possibility of future assay limitations once more MβL types or newly emerging MβL allelic variants are detected, requiring a possible assay reconfiguration.

The assay was able to detect and identify all MβL-harboring strains evaluated. It is a single-tube reaction, technically simple, performed in only 2 h after colony selection. The Tm-assigned MβL genotypes are easily interpreted (Fig. 1a) and may be suitable for the detection of MβL-producing gram-negative bacteria by molecular diagnostic laboratories. Furthermore, the assay may also be performed through a conventional amplification reaction, followed by visualization of the amplicons by using a UV light box after electrophoresis on a 1.5% agarose gel containing 0.5 μg/ml ethidium bromide (Fig. 1b).

The rapid detection of MβL-producing isolates could be helpful for epidemiological purposes and for monitoring the emergence of MβL-producing isolates in clinical settings. The detection of such isolates could help rapidly establish standards for hospital infection control measures to minimize the spreading of these resistant determinants.

ACKNOWLEDGMENTS

We thank Timothy R. Walsh, Mark A. Toleman, Yoshichika Arakawa, and Yunsop Chong for providing some of the MβL-harboring clinical isolates included in this study.